Eccles:Making Primary Cells from Tissue

Making Primary Cells from Mouse Tissue

Materials

Sterile Gem blades (wrap gem blades in tinfoil and autoclave)
Petri dishes
1x PBS
Ice
Sterile eppendorf tubes
Eppendorf tubes containing 1mL 1x PBS on ice - sterile (or larger tubes and PBS volume if you are making cells from large tissue samples - 15ml tubes)
Cell culture media without additives, ice cold (no FCS etc as this may interfere with collagenase type II activity)
Cell culture media with additives, ice cold and warmed aliquots (research suitable media for your tissue type)
Collagenase type II
Collagen coated cell culture vessels (a T25 flask was suitable for an embryonic mouse kidney ~0.5mg tissue and chunks of human ADPKD kidney of about 0.5mg)
Dry ice (small handful to euthanise your animal)
Dissection tools soaking in 70% EtOH
Euthanisation chamber (Cherie's bench)

Cell Culture Media used for mouse embryonic kidneys (no additives)

DMEM/F-12 (1:1) Invitrogen #113330-032, 500mL
Aliquot a working volume (usually 50mL) into 50mL tube
Store at 4 degrees C

Cell Culture Media used for mouse embryonic kidneys (with additives)

Reagent	50mL	500mL	Final Concentration
DMEM/F-12 (1:1)	46.85mL
insulin-transferrin-selenium (100x)	375uL	3.75mL	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	2.5mL	25mL	5%

Insulin-Transferrin-Selenium (100x)

Insulin-transferrin-selenium, 10mL, Invitrogen #41400-045
Store at 4 degrees C
Ready to use

Dexamethasone (20ug/mL)

Dexamethasone, 1mg, Sigma #D8893
Lyophilised reagent stored at 4 degrees C

• 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 and store at -20
• 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 and store at -20
• 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 6uL lots to avoid freeze/thawing
• Store at -20
• 0.1mg/mL stock solution is stable for up to 2 weeks at 4 degrees C once made up
• Use between 2-20ng/mL final (1-10ul in 50mL media)

Interferon-gamma, mouse recombinant

0.1mg, Sigma #I4777

After contacting Sigma technical help I identified that this batch of interferon-gamma contained not less than 1x10⁶ units
It seems that with any new batch of IFN-gamma ordered in one would need to check this (details didn't come with IFN-gamma)

• Make 1mg/mL solution in 1x PBS (0.1mg in 0.1mL of 1x PBS). This gives a 1x10⁷U/mL solution.
• Further dilute in DMEM/F-12 (1:1) no additives containing 10% FCS to 1x10⁵U/mL.
• Aliquoted into 100uL lots and stored at -20.
• Each time a 1x10⁵U/mL. aliquot is thawed it will need to be further diluted 1/10 to 1x10⁴U/mL in DMEM/F-12 (1:1) no additives containing 10% FCS.
• Aliquot 1x10⁴U/mL interferon-gamma and store at -20 to prevent repeated freeze/thawing.
• Once thawed the interferon-gamma solution is stable for 1 week at 4 degrees C

Collagenase Type II Solution

Work out volume of collagenase type II solution required (depends on how many tissue samples and sizes you'll be harvesting)
Here's some examples below of tissue that primary cells have been successfully made from.
Any excess collagenase solution can be aliquoted and stored at -20 (avoid freeze/thawing more than 2x)

Tissue Sample	Collagenase Type II Solution	Average Weight of Tissue Sample	Volume Collagenase/Sample	Amount Collagenase/Sample
HsADPKD kidney chunks 18.7.05	1mg/mL	estimate 0.5g	15mL	30mg/g tissue
Mouse Embryonic Kidneys example 1	1mg/mL	~5mg each	0.2mL	40mg/g tissue
Mouse Embryonic Kidneys example 2	2mg/mL	~5mg each	0.1mL	40mg/g tissue

• Aim to use a final concentration of 40mg collagenase type II/g of tissue
  
• Weigh desired amount of collagenase type II into 15ml tube and store on ice
  
• In cell culture hood aliquot volume of cell culture media (no additives) required and incubate on ice
  
• Add suitable volume of media to collagenase to give 1mg/mL or 2mg/mL solution and pipette up and down to resuspend
  
• Filter sterilise collagenase type II solution in cell culture hood
  
• Leave on ice until ready to use
  

Euthanising Animal

• Pour a little bit of water into bottom of chamber
• Add a few pellets of dry ice
• Place lid on chamber and swirl to get good gas emission
• Place mouse on centre platform and place lid on chamber
• Wait for 1min
• Check animal is dead
• Remove animal from chamber and tape onto dissection tray in a position suitable for collecting the desired tissue
• Squirt dissection area of mouse with 70% EtOH
• Begin dissection
• Remove desired tissue with sterile tools and place in tubes on ice containing 1x PBS
• Incubate on ice until ready to proceed

Recovering Cells from Tissue

• Take tissue on ice to cell culture hood
• Remove tissue from tube into sterile petri dish. Pipette on a small volume of 1x PBS to prevent tissue sticking to plate
• Use sterile Gem blade to chop tissue up using up and down chopping motions combined with a bit of smearing against dish surface. It is not necessary to completely mince up tissue
• Pipette the tissue soup into a fresh eppendorf
• Use ice cold media and pipette onto dish to wash up the last bits of tissue from plate - pipette into eppendorf (there will be a bit of tissue left behind which is fine)
• For embryonic mouse kidneys I've used 100uL of ice cold media without additives. I've then added 0.2mg of collagenase type II (100uL of 2mg/mL or 200uL of 1mg/mL). A final volume of 200uL or more worked well.
• Digest tissue at 37 degrees C for 30mins with shaking (850rpm on eppendorf shaker)
• Pellet tissue/cell debris at 800g, 5min
• Discard supernatant and resuspend tissue/cell debris in 1mL of media (with additives)
• Pellet tissue/cell debris at 800g, 5min
• Discard supernatant and bring tissue/cell debris up in warm cell culture media (with additives). I used 0.5mL for embryonic mouse kidneys
• Pipette tissue/cell debris in warmed media into collagen coated cell culture vessel of suitable size containing media and disperse as normally would. For the mouse embryonic kidneys I pipetted each half of the cell debris in media (250uL) onto one well of a 6 well plate (ended up with 2 wells of a 6 well plate for each kidney). We found this to be of suitable size and also allowed cloning of individual cells using cloning rings.
• Incubate at 37 degrees C, 5% CO₂
• Check cells each day
• Change media every 2nd day

For SV40 large T Positive Cells

• Once cells are established they can be moved to the permissive temperature of 33 degrees C, 5% CO₂
• Add interferon-gamma to a final concentration of 10units/mL in cell culture media.

Cell Culture Vessel	Volume of stock IFN-gamma to add (1x104U/mL)	Final [IFN-gamma]	Media volume
24 well plate	5ul of 1/10 dilution of stock IFN-gamma	10U/mL	0.5mL
6 well plate	2uL	10U/mL	2mL
T25 flask	5uL	10U/mL	5mL
T75 flask	25uL	10U/mL	25mL
T175 flask	50uL	10U/mL	50mL