Eccles:Making Primary Cells from Surgery Tissue

Making Primary Cells from Surgery Tissue

Materials

• Tubes of suitable size on ice containing enough media to submerge tissue (can collect tissue in either ice cold cell culture media suitable for tissue type or something like PBS, HBSS)
• Sterile Gem blades
• Sterile petri dishes
• Collagenase type II (1-2mg/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 human ADPKD kidney chunks I used T25 flasks)

To help you make some decisions about what might be suitable this is what we did to make primary cells from chunks of a human ADPKD kidney that I estimated as weighing ~0.5g each:

• 50mL tubes on ice containing 20mL cell culture media (DMEM/F-12 1:1, no additives)

Grow Cells on Collagen Coated Surfaces

Refer to (Collagen Coating of Cell Culture Vessels) for protocol.

Cell Culture Media Tissue Collected in (no additives)

DMEM/F-12 (1:1)

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	3.75mL	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)

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-50mL 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
• For the human ADPKD kidney tissue there were 9 samples of ~0.5g each. I made 150mL of 1mg/mL solution of collagenase type II and used 15mL per sample

Method

• Tissue was cut using sterile tools and placed into tube containing ice cold media
• Tissue was kept on ice until ready to proceed with harvesting cells
• Because I had other samples to process these tissue samples were actually incubated in their tubes (still in DMEM/F-12 1:1, no additives) at 37 degrees C overnight in cell culture incubator until I had time to harvest the cells
• 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)
• Pipette tissue soup into 15mL tube. Use ice cold media to wash up as much of tissue from petri dish as you can
• Add 40mg/g tissue of collagenase type II solution (for ~ 0.5g human ADPKD kidney chunks I added 15mL of 1mg/mL solution which ended up being 30mg/g tissue which worked fine)
• Incubate at 37 degrees C, 30mins with shaking
• Pellet tissue/cell debris at 250g, 5min (still in 15mL tubes)
• Resuspend tissue/cell debris in ice cold 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 (see above for media used for human ADPKD kidney chunks)
• Plate tissue/cell debris into collagen coated cell culture vessels containing warmed media
• Incubate at 37 degrees C, 5% CO₂
• Check cells daily and change media as necessary.
• Split cells as usual
• Continue to grow cells on collagen coated vessels