Eccles:HuADPKD Cell Culture

Background

These cells were made from ~0.5g chunks of kidney taken from a male with ADPKD.
Refer to Making Primary Cells From Surgery Tissue There were 9 different tissue samples in total. Which part of the kidney the tissue was taken from is unknown as the kidney was so riddled with cyts and fibrotic tissue that the original kidney anatomy was unrecognisable.
Six samples successfully grew in culture. Samples are identified as "sample 1" etc.

Grow Cells on Collagen Coated Surfaces

Refer to Collagen Coating of Cell Culture Vessels for protocol.

Cell Culture Media used for human ADPKD kidney cells (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%

• Grow cells at 37 degrees C, 5% CO₂.

• Trypsinise cells as usual.

• Split 1:6 maximum.

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)