Establishing a Primary Culture

1. Establishing a Primary Culture Skeletal Muscle

2. 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)

3. Considerations • Highly structured tissue • disaggregation • Heterogeneous • purification • Multi-cellular versus single cell • media • Contamination • Sterile technique • Proliferation versus differentiation • Media • substrate

4. 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

5. Laminar Flow Hood

6. Aseptic Technique • hair is tied • back • container for • disposal of • used tips • contains any • hazardous • aerosols in • the biohazard • cabinet

7. 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.

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

9. “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.

10. 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

11. 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)

12. Myogenesis

13. Collection • Sterility • Wash in 70% ethanol • Gentamicin • Pen/strep • (fungizone) • Speed • 45 minutes • Tissue collected/clean • Removal of tendon, fat, nerve

14. Disaggregation • Mechanical mincing • scissors • Collagenase • Dispase • Trypsin • Washing

15. Filtering • 100 micron • Removal of undigested material • Lets through single cells • Can count cells using haemocytometer to plate at a particular cell density

16. Cell Counting - haemocytometer

17. 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

18. 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)

19. 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

20. CO2 Incubator • Controlled CO2 • Humidified • 37oC

21. Inverted Microscope

22. Analysis

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

24. Fusion • Media changed from nutrient rich to nutrient poor • Induces withdrawal from the cell cycle giving the cells 3 choices • Die (apoptosis) • Senesce • Differentiation

25. Adult Mouse Skeletal Muscle - Primary culture cultured (on fibronectin) in 8 well slide, fixed and stained for desmin

26. Mouse Myogenic Cell line (H-2Kb) cultured (on Matrigel) in 8 well slide, fixed and stained for desmin

27. 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

28. Housekeeping/Maintenance • Proliferation media • Trypsinisation and splitting/passaging of cultures • Contact inhibition • 1% gelatin coated dishes • Cryopreservation • 10% FCS and 10% DMSO