Establishing a Primary Establishing a Primary CultureCulture

Skeletal MuscleSkeletal 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 grown in culture for 7 days.

Culture viewed on an inverted phase microscope. (phase microscopy)

ConsiderationsConsiderations

Highly structured tissueHighly structured tissue disaggregationdisaggregation

HeterogeneousHeterogeneous purificationpurification

Multi-cellular versus single cellMulti-cellular versus single cell mediamedia

ContaminationContamination Sterile techniqueSterile technique

Proliferation versus differentiationProliferation versus differentiation MediaMedia substratesubstrate

From a Solid Tissue Solid Tissue - need to separatecells 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

Primary Cell Culture Isolation

From a cell suspension tissue cell suspension tissue - blood or body fluid (ascites)• isolation by centrifugation (differential)• grown in large volume of tissue culture medium

Laminar Flow HoodLaminar Flow Hood

• hair is tied back

• container for disposal of used tips contains any hazardous aerosols in the biohazard cabinet

Aseptic Technique

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 plateAllows comparison of many culture conditions.Samples often in triplicate.

Pipettes (glass or disposable)are plugged to minimise aerosol contamination, whensolutions are expelled from them.

“Pipette-Aid” - power or battery operated

Motorised intake and expellingof 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.

Clarification

Microfiltration

Ultrafiltration

Reverse Osmosis

Micron = 10 -6 m

80

40

20

10

5

.8

.4

.2

.1

.05

2

.008

.004

.002

.001

.02

.0003

Human Hair DiameterSmallest Visible Particle

Erythrocyte

BacteriaMycoplasma

Polio Virus

S I Z E

0.8 pre-filter0.22 end filter 0.1

Tissue culture medium cannot be autoclaved. It is filtered through 0.2 membrane filters.

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

NOW many items are purchased sterile (expensive)

MyogenesisMyogenesis

CollectionCollection

SterilitySterility Wash in 70% ethanolWash in 70% ethanol GentamicinGentamicin Pen/strepPen/strep (fungizone)(fungizone)

SpeedSpeed 45 minutes45 minutes

Tissue collected/cleanTissue collected/clean Removal of tendon, fat, nerveRemoval of tendon, fat, nerve

DisaggregationDisaggregation

Mechanical mincingMechanical mincing scissorsscissors

CollagenaseCollagenase

DispaseDispase

TrypsinTrypsin

WashingWashing

FilteringFiltering

100 micron100 micron Removal of undigested materialRemoval of undigested material Lets through single cellsLets through single cells

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

Cell CountingCell Counting - haemocytometer - haemocytometer

Filtering and Pre-plateFiltering and Pre-plate

100 micron100 micron Removal of undigested materialRemoval of undigested material Lets through single cellsLets through single cells Can count cells to plate at a particular densityCan count cells to plate at a particular density

OR

Pre-platePre-plate 60 mins60 mins Differential attachment to culture plasticDifferential attachment to culture plastic

Skeletal MuscleFrom Mouse

Supernatant Transferred from flask to new flask after given time

Time0 60min

Putative Muscle DerivedStem Cell (MDSC)

etc

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

Media FormulationMedia Formulation

Proliferation/maintenanceProliferation/maintenance Hams F10 nutrient mixHams F10 nutrient mix 20% FCS (foetal calf serum)20% FCS (foetal calf serum) 5ng/ml bFGF5ng/ml bFGF

Differentiation and fusionDifferentiation and fusion DMEMDMEM 20% horse serum (Note: change in serum type)20% horse serum (Note: change in serum type) InsulinInsulin Linoleic aciudLinoleic aciud

COCO22 Incubator Incubator

Controlled COControlled CO22

HumidifiedHumidified

3737ooCC

Inverted MicroscopeInverted Microscope

AnalysisAnalysis

Primary muscle culture stained with desmin (green) – to identify myoblasts and Hoescht (blue) to identify cell nuclei

FusionFusion

Media changed from nutrient rich to Media changed from nutrient rich to nutrient poornutrient poor Induces withdrawal from the cell cycle giving Induces withdrawal from the cell cycle giving

the cells 3 choicesthe cells 3 choicesDie (apoptosis)Die (apoptosis)

SenesceSenesce

DifferentiationDifferentiation

Adult Mouse Skeletal Muscle - Primary culturecultured (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/MaintenanceHousekeeping/Maintenance

Proliferation mediaProliferation media

Trypsinisation and splitting/passaging of culturesTrypsinisation and splitting/passaging of cultures Contact inhibitionContact inhibition

1% gelatin coated dishes1% gelatin coated dishes

CryopreservationCryopreservation 10% FCS and 10% DMSO10% FCS and 10% DMSO