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  • Lecture 1 Intro To Tissue Culture

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    yeker55835
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    This document outlines the key steps and considerations for tissue culture methodology. It discusses the historical context of tissue culture techniques and how antibiotics and laminar flow hoods enabled the field to grow. The document then covers topics like primary cells versus cell lines, acquiring and processing tissue samples, quantifying and culturing cells, cryopreservation, and ensuring sterile conditions to prevent contamination. The overall conclusion is that tissue culture training is an essential skill that requires significant resources and commitment from universities to establish proper facilities and provide hands-on practical experience.

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    Lecture 1 Intro to Tissue Culture

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    This document outlines the key steps and considerations for tissue culture methodology. It discusses the historical context of tissue culture techniques and how antibiotics and laminar flow hoods enabled the field to grow. The document then covers topics like primary cells versus cell lines, acquiring and processing tissue samples, quantifying and culturing cells, cryopreservation, and ensuring sterile conditions to prevent contamination. The overall conclusion is that tissue culture training is an essential skill that requires significant resources and commitment from universities to establish proper facilities and provide hands-on practical experience.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    5 views19 pages

    Lecture 1 Intro To Tissue Culture

    Uploaded by

    yeker55835
    This document outlines the key steps and considerations for tissue culture methodology. It discusses the historical context of tissue culture techniques and how antibiotics and laminar flow hoods enabled the field to grow. The document then covers topics like primary cells versus cell lines, acquiring and processing tissue samples, quantifying and culturing cells, cryopreservation, and ensuring sterile conditions to prevent contamination. The overall conclusion is that tissue culture training is an essential skill that requires significant resources and commitment from universities to establish proper facilities and provide hands-on practical experience.

    Copyright:

    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    of 19

    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

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