Eccles:MKS Epithelial Cell Culture (Lynn)

=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.