PCB 4023 Macrophage cell subculture 1 by 1O7900m

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									Cell Biology Lab
Cell subculture

    PCB 4023 L
    TA: Sushmita Mustafi
Objectives:
   What is cell culture
   Importance
   How to get cells for ex vivo culture
   Primary and secondary culture
   Physical/chemical requirements for cell culture
   Subculture [ splitting]
   Established cell lines
   J774-Eclone cells and their specifications
   3T3 cells
   How to work in Biosafety cabinet.
What is cell culture?

   Prokaryotic/ Eukaryotic cells are grown under
    controlled condition.

Why?

   Cellular and molecular studies.
   Protein over expression/ structure and function
    studies
   Drug development
   Vaccine test.
How to isolate cells to grow them under
controlled condition?
   Apart from blood …. cells are attached in
    tissue with the help of extracellular matrix.

   Different enzymes like Trypsin/ pronase/
    collagenase are used to break down the
    matrix and isolate the cells
   Explant culture: Harvesting tissue to collect
    cells.
Cell line can be two types

   PRIMARY CELL LINE             SECONDARY CELL LINE
   Directly from subject         From tumor / cancer cell line
   Finite, short life span.      Infinite/ can be stored and
                                   cultured for years.
   Very specific in              Major established cell lines,
    individual research labs       commercially available.
How to maintain cell lines
Physical condition……..
Incubator
                   -Provides the ideal
                     environment for
                     cells.
                   -Work to control 3
                     essential variables.
                   (1)Temperature: 37°C
                   (2)CO2 content: 5% CO2
                   (3)Humidity: 95%
                    (4) pH – 7.2 to 7.5
Chemical conditions:
Media                -   Liquid designed to
                         support the growth of
                         your cells.

                     -   There are different
                         types of media for
                         different types of
                         cells.
                     -   AMEM
                     -   DMEM
                     -   RPMI
What's in that media?

    Growth medium:
    1) Bulk ions [ Sodium/ potassium/ calcium/
     Magnesium etc.]
    2)Trace element [ Iron/ Zn/ Se].
    3) Sugar.
    4) Amino acids.
    5) Vitamins.
    6) Choline/ inositol.
    7) Serum with growth hormones.
    8) Antibiotics.
Working with cell lines
What do we need?
Cell Line
Flask
Media
incubator
Microscope
Biological Safety Cabinet (Hood)
Flasks and Plates
Confluency?

   Approximately the amount of space inside the
    tissue culture flask being covered by cells.
   Expressed as %, like 50% confluency.
   Cell growth and increase in number
    increases confluency
   High confluency could be harmful for cells.
Why?

   Accumulation of apoptotic/ necrotic cells
   Cell to cell contact stimulate cell cycle arrest
What to do?
Split cells….
 Add Trypsin / using pipette of hitting the flask
 detach cells from the adherent surface of flask.
 Use a new flask

 Add fresh media to the new flask

 Introduce cells to the new flask with fresh media in
  5:1 ratio.
 Each time you split, you are making a new
  passage… u should keep a note of passage
  number. Label them as P1/P2 etc..
 Why is it important????
Some important cell line

   Human
   Hela - Cervical cancer
   MCF-7 – Breast cancer

   Mouse
   3T3-L1 [ fibroblast]
   RAW
   J774-Eclone
Our cell line

  J774-Eclone : Mouse macrophage cell line.
 Popular in phagocytosis studies and other immunological
   studies.
 Media Used: DMEM with 10% FBS, 1% non essential
   amino acid, 1% sodium Pyruvate,
  1% pen/strep.
3T3L1

   3T3-L1
   Mouse embryonic fibroblast- adipose like cell
    line.
   Derived from swiss 3T3 mouse.
   Used as pre adiposites
   Media: DMEM with FBS.
Biological Safety Cabinets
Leak-tight
construction, HEPA
filters, and airflow
patterns work to
protect product,
personnel, or both.
Inverted Microscope

- Light source and
   condenser are on the
   top of the stage, while
   the objectives and turret
   are below.
-Allows for better
   observation of living
   cells at the bottom of a
   large container
Contamination

								
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