Eccles:MKS Epithelial Cell Culture (Lynn)

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Making Primary Cells from MKS3 Sheep Tissue

Materials

  • 50 ml tubes on ice with 10ml of media (can collect tissue in either ice cold cell culture media, PBS or HBSS)
  • Sterile Gem blades
  • Sterile petri dishes
  • Collagenase type I (10mg/ml solution)
  • Cell culture media (no additives)
  • Cell culture media (with additives)
  • Collagen coated cell culture vessel of suitable size for tissue sample (for ~0.5g kidney chunks use T75 flasks):


Method

  • NB coat flask for at least 1 hour before plating out cells
  • Cut tissue using sterile tools and place into tube containing ice cold media no additives
  • Can leave tissue on ice until ready to proceed with harvesting cells or place at 370 C overnight in cell culture incubator
  • In cell culture hood remove tissue into a sterile petri dish
  • Using sterile Gem blade chop tissue up until it resembles a tissue soup (some small chunks remain which is fine) NB cystic kidney is difficult to cut however normal kidney disintegrates easily
  • Pipette tissue soup into 50ml tube. Use media to wash up as much of tissue from petri dish as you can
  • For ~ 0.5g tissue use 15ml of media at 1mg/ml (this is ~30mg/g tissue), add 1.5 ml of a 10mg/ml stock to tissue/media & make to a final volume of 15ml with media
  • Incubate at 370 C, 30mins with shaking orbital shaker, 125rpm (Robertson’s)
  • Pellet tissue/cell debris at 250g, 5min
  • Resuspend tissue/cell debris in warm cell culture media (no additives)
  • Repeat last two steps once more to wash away residual collagenase solution
  • Pellet tissue/cell debris at 250g, 5min
  • Resuspend in warm cell culture media containing necessary additives for growth. Remove the collagen from the flask & add tissue/cell debris into the flask
  • Incubate at 370 C, 5% CO2
  • After 24 hours remove the non-adherent cells into a fresh collagen coated flask, gently washing the flask with media. (It is possible to spin at 250g for 5 minutes & resuspend in fresh media).
  • Check cells daily and change media as necessary
  • Split cells as usual
  • Continue to grow cells on collagen coated vessels


Cell Culture Media Cells Grown in (with additives)

Reagent 50ml 500ml Final Concentration DMEM/F-12 (1:1) 46.85mL insulin-transferrin-selenium (100x) 375ul 3.75mlL 0.75x dexamethasone (20ug/ml) 250ul 2.5ml 100ng/ml triiodotyronine (20ug/ml) 15ul 150ul 6ng/ul murine EGF (0.1mg/ml) 5ul 50ul 10ng/ml pen/strep 0.5ml 5ml FCS 2.5mlL 25ml 5%

Insulin-Transferrin-Selenium (100x)

Insulin-transferrin-selenium, 10mlL, Invitrogen #41400-045

 Store at 40C 
Ready to use [fridge rm 210]

Dexamethasone (20ug/ml)

Dexamethasone, 1mg, Sigma #D8893

 Dissolve 1mg dexamethasone in 1ml of absolute ethanol to give a 1mg/ml solution
Dilute 1 in 50 by adding 49ml media (DMEM/F-12 1:1 without additives) to give a final stock concentration of 20ug/ml
Aliquot into 500ul & 1ml aliquots and store at -200C [freezer rm 210]
Use between 20-200ng/ml final (50-500ul in 50ml media)


Triiodotyronine (20ug/ml)

Triiodotyronine, 1mg, Sigma #T5516
Dilute 1mg in 1ml sterile 1M NaOH
Dilute 1 in 50 by adding 49ml media (DMEM/F-12 1:1 without additives) to give a final stock concentration of 20ug/mL
Aliquot into 15 & 150ul aliquots and store at -200C [freezer rm 210]
Use between 0.6-6ng/ml final (1.5-15ul in 50ml media)


Murine Epidermal Growth Factor

mEGF, 0.1mg, Sigma #E4127
Add 1ml media containing 10% FCS (0.9ml DMEM/F-12 1:1 without additives + 0.1ml FCS)
Aliquot into 55ul & 6ul aliquots to avoid freeze/thawing
Store at -200C [freezer rm210]
0.1mg/mL stock solution is stable for up to 2 weeks at 40C once made up
Use between 2-20ng/ml final (1-10ul in 50ml media)


Collagenase Type I Solution

Sigma C9891-500mg
Make a 10mg/ml solution in media. Resuspend by pipetting up & down gently
Spin at 300xg for 5 minutes to pellet insoluble debris
Filter sterilze (0.2um) & aliquot into 1.5ml & 1ml aliquots. Store at -200C [freezer rm210]
NB this solution is difficult to filter !
(Collagenase Type IV Sigma C-5138-500mg)

Collagen Type IV Solution

Sigma C7521-10mg
Make a 1mg/ml solution in 0.1M acetic acid (57.2ul 100% acetic acid plus 9.943 ml water), dissolve for several hours at 40C, occasionally swirling
Filter sterilze
Dilute collagen in 1x PBS to 0.05ug/ul ready for use (store at 40C. [2.55ml stock plus 47.45ml PBS])
Stock solution in the fridge rm 210, working stock in the fridge in rm 203
(Collagen Type I Sigma C9791)

Collagen Coating of Cell Culture Vessels

Pipette collagen solution (0.05ug/ul collagen in 1x PBS) onto surface and swirl to disperse as you would with cells.
Leave collagen solution on plate surface for 2h (can be left in cell culture hood or incubator).
Aspirate off liquid. Add media to keep collagen moist. Add cells as usual or vessel can be kept in cell culture incubator until ready to add cells.
Vessel Size Surface Area (cm2) Media Volume Volume Collagen Solution 96 well plate 0.28 0.2ml 33ul 24 well plate 2.0 0.5ml 300ul 6 well plate 

35x10mm dishes	9.6	2ml	1.1ml

T25 flask 25 5ml 2.9ml T75 flask 75 20ml 8.8ml T175 flask 175 50ml 20.6ml

Freezing Media

65% DMEM/F12 (no additives)
25% FCS
10% DMSO
Mix & filter sterilze.

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