Establishing a primary culture
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Establishing a Primary Culture. Skeletal Muscle. Muscle regeneration in vivo - likeness to formation of myotubes in culture. Haematoxylin and Eosin stained paraffin embedded section of mouse muscle 12 days after injury (viewed by bright field microscopy). Myotubes formed by myoblasts

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Muscle regeneration in vivo

- likeness to formation of

myotubes in culture.

Haematoxylin and Eosin stained

paraffin embedded section of

mouse muscle 12 days after injury

(viewed by bright field microscopy)

Myotubes formed by myoblasts

grown in culture for 7 days.

Culture viewed on an inverted

phase microscope.

(phase microscopy)


Considerations
Considerations

  • Highly structured tissue

    • disaggregation

  • Heterogeneous

    • purification

  • Multi-cellular versus single cell

    • media

  • Contamination

    • Sterile technique

  • Proliferation versus differentiation

    • Media

    • substrate


Primary Cell Culture Isolation

  • From a Solid Tissue - need to separate

  • cells of interest from connective tissue

  • and extra cellular matrix

  • explant culture

  • mechanical and enzymic dissociation

  • washed in medium with serum

  • filtered and plated in final medium

  • From a cell suspension tissue - blood or body fluid (ascites)

  • isolation by centrifugation (differential)

  • grown in large volume of tissue culture medium



Aseptic

Technique

  • hair is tied

  • back

  • container for

  • disposal of

  • used tips

  • contains any

  • hazardous

  • aerosols in

  • the biohazard

  • cabinet


8 well culture dish.

Allows comparison of 8 samples:

can have different stains or

are fixed at different times.

THEN- remove wells and gasket.

Leaves ONE slide with 8

separate samples for easy

microscopic analysis

of (stained) cells

96 well plate

Allows comparison of many

culture conditions.

Samples often in triplicate.


Pipettes (glass or disposable)

are plugged to minimise

aerosol contamination, when

solutions are expelled from

them.


“Pipette-Aid”

- power or battery operated

Motorised intake and expelling

of fluids transferred from one

sterile container to another.

In line air filter.

“Transfer Pipette”

Disposable Enclosed Plastic

Packaged as Sterile so their

contained air is sterile.


80

40

20

10

5

2

.8

.4

.2

.1

.05

.02

.008

.004

.002

.001

.0003

S I Z E

Human Hair Diameter

u

u

Smallest Visible Particle

Clarification

Micron = 10 -6 m

Erythrocyte

u

Microfiltration

Bacteria

u

0.8m pre-filter

0.22m end filter

0.1 m

Mycoplasma

u

Polio Virus

u

Ultrafiltration

Reverse Osmosis


Tissue culture medium cannot be autoclaved.

It is filtered through 0.2m membrane filters.

There are different filter membrane types for sterilizing gases, solvents and aqueous solutions.

NOW many items are purchased sterile (expensive)



Collection
Collection

  • Sterility

    • Wash in 70% ethanol

    • Gentamicin

    • Pen/strep

    • (fungizone)

  • Speed

    • 45 minutes

  • Tissue collected/clean

    • Removal of tendon, fat, nerve


Disaggregation
Disaggregation

  • Mechanical mincing

    • scissors

  • Collagenase

  • Dispase

  • Trypsin

  • Washing


Filtering
Filtering

  • 100 micron

    • Removal of undigested material

    • Lets through single cells

    • Can count cells using haemocytometer to plate at a particular cell density


Cell counting haemocytometer
Cell Counting - haemocytometer


Filtering and pre plate
Filtering and Pre-plate

  • 100 micron

    • Removal of undigested material

    • Lets through single cells

    • Can count cells to plate at a particular density

      OR

  • Pre-plate

    • 60 mins

    • Differential attachment to culture plastic


Pre plating to remove other cells e g fibroblasts
Pre-plating – to remove other cells, e.g fibroblasts

Time

Skeletal Muscle

From Mouse

etc

0

60min

Supernatant Transferred from flask to new flask after given time

Putative Muscle Derived

Stem Cell (MDSC)


Media formulation
Media Formulation

  • Proliferation/maintenance

    • Hams F10 nutrient mix

    • 20% FCS (foetal calf serum)

    • 5ng/ml bFGF

  • Differentiation and fusion

    • DMEM

    • 20% horse serum (Note: change in serum type)

    • Insulin

    • Linoleic aciud


Co 2 incubator
CO2 Incubator

  • Controlled CO2

  • Humidified

  • 37oC




Primary muscle culture stained with

desmin (green) – to identify myoblasts and

Hoescht (blue) to identify cell nuclei


Fusion
Fusion

  • Media changed from nutrient rich to nutrient poor

    • Induces withdrawal from the cell cycle giving the cells 3 choices

      • Die (apoptosis)

      • Senesce

      • Differentiation


Adult Mouse Skeletal Muscle - Primary culture

cultured (on fibronectin) in 8 well slide,

fixed and stained for desmin


Mouse Myogenic Cell line (H-2Kb)

cultured (on Matrigel) in 8 well slide,

fixed and stained for desmin


Mouse Skeletal Muscle Cell Line (H-2Kb)

Proliferation

Scattered myoblasts

at time of subculture.

Differentiation and fusion

Myotubes formed at 5 days after

transfer of near confluent culture

to fusion medium


Housekeeping maintenance
Housekeeping/Maintenance

  • Proliferation media

  • Trypsinisation and splitting/passaging of cultures

    • Contact inhibition

  • 1% gelatin coated dishes

  • Cryopreservation

    • 10% FCS and 10% DMSO


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